Method for detection of drug-induced mutations in the reverse transcriptase gene

ABSTRACT

The present invention relates to a method for the rapid and reliable detection of drug-induced mutations in the reverse transcriptase gene allowing the simultaneous characterization of a range of codons involved in drug resistance using specific sets of probes optimized to function together in a reverse-hybridization assay. More particularly, the present invention relates to a method for determining the susceptibility to antiviral drugs of HIV strains present in a biological sample, comprising: (i) if need be releasing, isolating or concentrating the polynucleic acids present in the sample; (ii) if need be amplifying the relevant part of the reverse transcriptase genes present in said sample with at least one suitable primer pair; (iii) hybridizing the polynucleic acids of step (i) or (ii) with at least two RT gene probes hybridizing specifically to one or more target sequences with said probes being applied to known locations on a solid support and with said probes being capable of simultaneously hybridizing to their respective target regions under appropriate hybridization and wash conditions allowing the detection of homologous targets, or said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence wherein T is replaced by U; (iv) detecting the hybrids formed in step (iii); (v) inferring the nucleotide sequence at the codons of interest and/or the amino acids of the codons of interest and/or antiviral drug resistance spectrum, and possibly the type of HIV isolates involved from the differential hybridization signal(s) obtained in step (iv).

This is a divisional of application Ser. No. 08/913,833, filed Sep. 15, 1997, U.S. Pat. No. 6,087,093, which is herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of HIV diagnosis. More particularly, the present invention relates to the field of diagnosing the susceptibility of an HIV sample to antiviral drugs used to treat HIV infection.

2. Description of the Related Art

The present invention relates to a method for the rapid and reliable detection of drug-induced mutations in the HIV reverse transcriptase gene allowing the simultaneous characterization of a range of codons involved in drug resistance using specific sets of probes optimized to function together in a reverse-hybridisation assay.

During the treatment of human immunodeficiency virus (HIV) type 1 infected individuals with antiretroviral nucleoside analogs emergence of resistance against these drugs has been observed. The mechanism responsible for the resistance is not fully understood, since the appearance of a resistant virus in not always correlated with clinical deterioration (Boucher et al. 1992). Amongst the reverse transcriptase (RT) inhibitors, the nucleoside analogs 3′-azido-2′,3′-dideoxyThymidine (AZT, Zidovudine), 2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxyCytidine (ddC), (−)-β-L-2′,3′-dideoxy-3′-thioCytidine (3TC), 2′,3′-didehydro-3′deoxyThymidine (D4T) and (−)-2′,3′-dideoxy-5-fluoro-3′-thiacytidine (FTC) are the most important, since they show a favourable ratio of toxicity for the host versus efficacy as antiviral. All these compounds act in a similar way, namely they serve, after intracellular phosphorylation, as chain terminators of the RT reaction. Upon prolonged treatment with these nucleoside analogs, accumulation of mutations in the viral reverse transcriptase gene (RT) occur, thereby escaping the inhibitory effect of the antivirals. The most important mutations induced by the above compounds and leading to gradually increasing resistance were found at amino acid (aa) positions 41 (M to L), 69 (T to D), 70 (K to R), 74 (L to V), 181 (Y to C), 184 (M to V) and 215 (T to Y or F) (Schinazi et al., 1994). Mutations at aa 65, 67, 75 and 219 have also been reported but these were only showing a minor decrease in sensitivity. More recently, multi-drug-resistant HIV-1 strains were described showing aa changes at codon 62, 75, 77, 116, and 151 (Iversen et al., 1996). In general, these aa changes are the consequence of single point mutations at the first or second codon letter, but in the case of T69D (ACT to GAT), T215Y (ACC to TAC) and T215F (ACC to TTC), two nucleotide mutations are necessarry. Whether in these cases the single nucleotide mutation intermediates exist, and if they show any importance in the mechanism for acquiring resistance is as yet not reported. Third letter variations are in general not leading to an amino acid change, and are therefore seen as natural polymorphisms.

The regime for an efficient antiviral treatment is not clear at all. The appearance of one or several of these mutations during antiviral treatment need to be interpreted in conjunction with the virus load and the amount of CD4 cells. Indeed, since it has been shown that the effect of AZT resistance mutations can be suppressed after the appeareance of the 3TC induced M184V mutation, it is clear that disease progression is multifactorial. The influence of other simultaneous occuring mutations under different combination therapies with respect to the outcome and resistance of the virus has not yet been analysed systematically. In order to get a better insight into the mechanisms of resistance and HIV biology, it is necessarry to analyse follow-up plasma samples of antiviral treated patients for these mutational events together with the simultaneous occuring changes of virus titre and CD4 cells.

SUMMARY OF THE INVENTION

It is an aim of the present invention to develop a rapid and reliable detection method for determination of the antiviral drug resistance of viruses which contain reverse transcriptase genes such as HIV retroviruses and Hepadnaviridae present in a biological sample.

More particularly it is an aim of the present invention to provide a genotyping assay allowing the detection of the different HIV RT gene wild type and mutation codons involved in the antiviral resistance in one single experiment.

It is also an aim of the present invention to provide an HIV RT genotyping assay or method which allows to infer the nucleotide sequence at codons of interest and/or the amino acids at the codons of interest and/or the antiviral drug resistance spectrum, and possibly also infer the HIV type or subtype isolate involved.

Even more particularly it is an aim of the present invention to provide a genotyping assay allowing the detection of the different HIV RT gene polymorphisms representing wild-type and mutation codons in one single experimental setup.

It is another aim of the present invention to select particular probes able to discriminate wild-type HIV RT sequences from mutated or polymorphic HIV RT sequences conferring resistance to one or more antiviral drugs, such as AZT, ddI, ddC, 3TC or FTC. D4T or others.

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV RT sequences from mutated or polymorphic HIV RT sequences conferring resistance to AZT.

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV RT sequences from mutated HIV RT sequences conferring resistance to ddI.

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV RT sequences from mutated HIV RT sequences conferring resistance to ddC.

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV RT sequences from mutated HIV RT sequences conferring resistance to 3TC.

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV RT sequences from mutated HIV RT sequences conferring resistance to D4T.

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV RT sequences from mutated HIV RT sequences conferring resistance to FTC.

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV RT sequences from mutated HIV RT sequences conferring resistance to multiple nucleoside analogues (i.e. multidrug resistance).

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV RT sequences from mutated HIV RT sequences conferring resistance to nevirapine.

It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV RT from mutated HIV RT sequences involving at least one of amino acid positions 41 (M to L), 50 (I to T), 67 (D to N), 69 (T to D), 70 (K to R), 74 (L to V), 75 (V to T), 151 (Q to M or L), 181 (Y to C), 184 (M to V), 15 (T to Y or F) and 219 (K to Q or E) of the viral reverse transcriptase (RT) gene.

It is particularly an aim of the present invention to select a particular set of probes, able to discriminate wild-type HIV RT sequences from mutated HIV RT sequences conferring resistance to any of the antiviral drugs defined above with this particular set of probes being used in a reverse hybridisation assay.

It is moreover an aim of the present invention to combine a set of selected probes able to discriminate wild-type HIV RT sequences from mutated HIV RT sequences conferring resistance to antiviral drugs with another set of selected probes able to identify the HIV isolate, type or subtype present in the biological sample, whereby all probes can be used under the same hybridisation and wash-conditions.

It is also an aim of the present invention to select primers enabling the amplification of the gene fragment(s) determining the antiviral drug resistance trait of interest.

The present invention also aims at diagnostic kits comprising said probes useful for developing such a genotyping assay.

All the aims of the present invention have been met by the following specific embodiments.

The present invention relates more particularly to a method for determining the susceptibility to antiviral drugs of an HIV retrovirus present in a biological sample, comprising:

(i) if need be releasing, isolating or concentrating the polynucleic acids present in the sample;

(ii) if need be amplifying the relevant part of the reverse transcriptase genes present in said sample with at least one suitable primer pair;

(iii) hybridizing the polynucleic acids of step (i) or (ii) with at least two RT gene probes hybridizing specifically to at least one target sequence as mentioned in any of figure land tables 1, 2 or 4, with said probes being applied to known locations of a solid support and with said probes being capable of simultaneously hybridizing to their respective target regions under appropiate hybridization and wash conditions allowing the detection of homologous targets, or with said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence wherein T in said target sequence is replaced by U;

(iv) detecting the hybrids formed in step (iii);

(v) and in most cases inferring the nucleotide sequence at the codons of interest and/or the amino acids at the codons of interest and/or the antiviral drug resistance spectrum, and possibly the type of HIV isolates involved from the differential hybridization signal(s) obtained in step (iv).

The relevant part of the RT gene refers to the regions in the RT gene harboring mutations causing resistance to antiviral drugs as described above and is particularly comprised between codons 1 and 241, and more particularly between codons 29 and 220 of the RT gene.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1H: Natural and drug induced variability in the vicinity of codon 41, 50, 67-70, 74-75, 150, 181-184, 215 and 219 of thr HIV RT gene. The most frequently observed wilder-type sequence is shown in the top line. Naturally occurring variations are indicated below. Drug-induced variants are indicated in bold italics.

FIG. 2A. Reactivities of the selected probes for codon 41 immobilized on LiPA strips with reference material. The positionn of each probe on the membrane strip is shown at the right of each panel. The sequence of the relevant part of the selected probes is give in Table 4. Each strip is indicated with a biotinylated PCR fragment from the reference panel. The reference panel accesion numbers are indicated in Table 4. For several probes multiple reference panel possibilities are available, but only one relevant acession number given each time. *: False positive reactivities. On top of the strips, the amino acids at the relevant codon, as derived from the probe reactivity, is indicated.

FIG. 2B. Reactivities of the selected probes for codons 69-70 immobilized on LiPA strips with reference material. The position of each probe on the membrane strip is shown at the right of each panel. The sequence of the relevant part of the selected probes is given in Table 4. Each strip is incubated with a biotinylated PCR fragment from the reference panel. The reference paenl accession numbers are indicated in Table 4. For several probes multiple reference panel possibilities are available, but only one relevant accession number given each time. *: False positive reactivities. On top of the strips, the amino acids at the relevant codon, as derived from the probe reactivity, is indicated.

FIG. 2C. Reactivities of the selected probes for codons 74-75 immobilized on LiPA strips with reference material. The position of each probe on the membrane strip is shown at the right ofeach panel. The sequence of the relevant part of the selected probes is given in Table 4. Each strip is incubated with a biotinylated PCR fragment from the reference panel. The reference panel accession numbers are indicated Table 4. For several probes multiple reference panel possibilities are available, but only one relevant accession number given each time. On top of the strips, the amino acids at the relevant codon, as derived from the probe reactivity, is indicated.

FIG. 2D. Reactivities of the selected probes for codon 184 immobilized on LiPA strips with reference material. The position of each probe on the membrane strip is shown at the right of each panel. The sequence of the relevant part of the selected probes is given in Table 4. Each strip is incubated with a biotinylated PCR fragment from the reference panel. The reference panel accession numbers are indicated in Table 4. FOr several probes multiple reference panel possibilities are available, but only one relevant accession number given each time. On top of the strips, the amino acids at the relevant codon, as derived from the probe reactivity, is indicated.

FIG. 2E. Reactivities of the selected probes for codon 215 immobilized on LiPA strips with reference material. The position of each probe on the membrane strip is shown at the right of each panel. The sequence of the relevant part of the selected probes is given in Table 4. Each strip is incubated with a biotinylated PCR fragment from the reference panel. The reference panel accession numbers are indicated in Table 4. For several probes multiple reference panel possibilities are available, but only one relevant accession number given each time. On top of the strips, the amino acids at the relevant codon, as derived from the probe reactivity, is indicated.

FIG. 2F. Reactivities of the selected probes for codon 219 immobilized on LiPA strips with reference material. The position of each probe on the membrane strip is shown at the right of each panel. The sequence of the relevant part of the selected probes is given in Table 4. Each strip is incubated with a biotinylated PCR fragment from the reference panel. The reference panel accession numbers are indicated in Table 4. For several probes multiple reference possibilities are available, but only one relevant accession number given each time. On top of the strips, the amino acids at the relevant codon, as derived from the probe reactivity, is indicated.

FIGS. 3A-3I. Clinical and virological features detectable in three patient follow-up samples. All three patients were infected with s HIV-1 strain showing the M41-T69-ZK70-L74-V75-M184-F214-T215-K219 genotype (wild type pattern). Top: Fluctuations between plasma HIV RNA copy numberd (▪) and CD4 cell count (x) are given in function of time. The different treatment regimens and the period of treatment is indicated on top. Middle: Changes that appeared during the treatment period and that could be scored by means of the LiPA probes are indicated, for patient 91007 at codon 41 and 215: for patient 94013 at codon 184: for patient 92021 at codon 70, 214, 215, 219. Bottem: Corresponding LiPA strips for a subset of the aa changes are shown. LiPA probes are indicated on the left, the as interpretation is indicated at the right of each panel.

FIG. 4. Reactivities of the selected probes for codons 151 and 181 on LiPA strips with reference material. The position of each probe on the membrane strip is shown at the right of each panel. The sequence of the relevant part of the selected probes given in Table 3. LiPA strips were incubated with sequence-confirmed PCR fragments, extracted and amplified from: a wild-type HIV-1 isolate (strip 1), a wild-type isolate with a polymorphism at codon 51 (strip 2) or 149 (strip 3), a multi-drug resistant HIV-1 isolate (strip 4) with no information about codon 181 and a non-nucleoside analogue treated HIV-1 isolate which remains wild-type at codon 151 (strip 5).

DETAILED DESCRIPTION OF THE INVENTION

According to a preferred embodiment of the present invention, step (iii) is performed using a set of at least 2, preferably at least 3, more preferably at least 4 and most preferably at least 5 probes meticulously designed as such that they show the desired hybridization results, when used in a reverse hybridisation assay format, more particularly under the same hybridization and wash conditions.

According to a preferred embodiment, the present invention relates to a set of at least 2 probes each targetting one or more of the nucleoside RT inhibitor induced nucleotide changes or target sequences including such a nucleotide change as indicated in any of FIG. 1 or Tables 1, 2 or 4. The numbering of HIV-1 RT gene encoded amino acids is as generally accepted in literature.

More prefererably, the present invention relates to a set of two or more probes each targetting two, three, four, five or more different nucleoside RT inhibitor induced nucleotide changes as indicated in any of FIG. 1 or Tables 1, 2 or 4.

More particularly, the present invention relates to a set of at least 2 probes allowing the characterization of a wild-type, polymorphic or mutated codon at any one of the drug-induced mutation positions represented in any of FIG. 1 or Tables 1 or 2 or at any one of the polymorphic positions represented in Table 4.

Even more particularly, the present invention relates to a set of at least 2 probes allowing the characterization of a wild-type, polymorphic or mutated codon at any of the positions represented in FIG. 1.

All the above mentioned sets of probes have as a common characteristic that all the probes in said set are designed so that they can function together in a reverse-hybridization assay, more particularly under similar hybridization and wash conditions. A particularly preferred set of probes selected out of the probes with SEQ ID NO 1 to 161 of Table 3 is described in example 2.2 and is indicated in Table 4 and FIG. 2. The particularly selected probes are also indicated in Table 3.

A particularly preferred embodiment of the present invention is a method for determining the susceptibility to antiviral drugs of an HIV isolates in a sample using a set of probes as defined above, wherein said set of probes is characterized as being chosen such that for a given mutation disclosed in any of FIG. 1, or Tables 1, 2 or 4 the following probes are included in said set;

at least one probe for detecting the presence of drug induced mutation at said position;

at least one probe for detecting the presence of a wild-type sequence at said position;

preferably also (an) additional probe(s) for detecting wild-type polymorphisms at positions surrounding the mutation position.

Inclusion of the latter two types of probes greatly contributes to increasing the sensitivity of said assays as demonstrated in the examples section.

A particularly preferred set of probes in this respect is shown in Tables 3 and 4 and FIGS. 2 and 3.

Selected sets of probes according to the present invention include at least one probe, preferably at least two probes, characterizing the presence of a drug-induced mutation in a codon position chosen from the following list of codons susceptible to mutations in the HIV RT gene: 41, 50, 67, 69, 70, 74, 75, 151, 181, 184, 215 or 219. Said probes being characterized in that they can function in a method as set out above.

Also selected probes according to the present invention are probes which allow to differentiate any of the nucleotide changes as represented in any of FIG. 1 or Tables 1, 2 or 4. Said probes being characterized in that they can function in a method as set out above.

Also selected sets of probes for use in a method according to the present invention include at least one, preferably at least two (sets of) probes, with said probes characterizing the presence of a drug-induced mutation in two codon positions chosen from the following list of codon combinations, with said codons being susceptible to mutations in the HIV RT gene: 41 and/or 50; 41 and/or 67; 41 and/or 69; 41 and/or 70; 41 and/or 74; 41 and/or 75; 41 and/or 151; 1 and/or 181; 41 and/or 184; 41 and/or 215; 41 and/or 219; 50 and/or 67; 50 and/or 69; 50 and/or 70; 50 and/or 74; 50 and/or 75; 50 and/or 75; 50 and/or 151; 50 and/or 181; 50 and/or 184; 50 and/or 215; 50 and/or 219; 67 and/or 69; 67 and/or 70; 67 and/or 74; 67 and/or 75; 67 and/or 151; 67 and/or 181; 67 and/or 184; 67 and/or 215; 67 and/or 219; 69 and/or 70; 69 and/or 74; 69 and/or 75; 69 and/or 151; 69 and/or 181; 69 and/or 184; 69 and/or 215; 69 and/or 219; 70 and/or 74; 70 and/or 75; 70 and/or 151; 70 and/or 181; 70 and/or 184; 70 and/or 215; 70 and/or 219; 74 and/or 75; 74 and/or 151; 74 and/or 181; 74 and/or 184; 74 and/or 215; 74 and/or 219; 75 and/or 151; 75 and/or 181; 75 and/or 184; 75 and/or 215; 75 and/or 219; 151 and/or 181; 151 and/or 184; 151 and/or 215; 151 and/or 219; 181 and/or 184; 181 and/or 215; 181 and/or 219; 184 and/or 215; 184 and/or 219; 215 and/or 219.

Even more preferred selected sets of probes for use in a method according to the present invention include in addition to the probes defined above a third (set of) probe(s) characterizing the presence of a third drug-induced mutation at any of positions 41, 50, 67, 69, 70, 74, 75, 151, 181, 184, 215 or 219, or particular combinations thereof.

Particularly preferred is also a set of probes which allows simultaneous detection of antiviral resistance at codons 41, 50, 67, 69, 70, 74, 75, 151, 181, 184 and 215, possibly also at codon 219.

An additional embodiment of the present invention includes at least one probe, preferably at least two probes, characterizing the presence of a drug-induced mutation in codon positions chosen from the list of codons susceptible to mutations in the HIV RT gene as mentioned in any of Table 1 or 2, such as at codons 65, 115, 150, 98, 100, 103, 106, 108, 188, 190, 138, 199, 101, 179, 236, 238 or 233, with said probes forming possibly part of a composition.

Particularly preferred embodiments of the invention thus include a set of probes for codon 41 comprising at least one, preferably at least two, probe(s) for targetting at least one, preferably at least two, nucleotide chances in any the following codons as represented in region I in FIG. 1:

wild-type codon E40 (GAA) or polymorphic codon E40 (GAG), mutant codon L41 (TTG) or L41 (CTG) or wild-type codon M41 (ATG), wild-type codon E42 (GAA) or polymorphic codon E42 (GAG), wild-type codon K43 (AAG) or polymorphic codon K43 (AAA) or polymorphic E43 (GAA).

Particularly preferred embodiments of the invention thus include a set of probes for codon 50 comprising at least one, preferably at least two, probe(s) for targetting at least one, preferably at least two, nucleotide chances in any the following codons as represented in region II in FIG. 1:

wild-type codon K49 (AAA) or polymorphic codon R49 (AGA), mutant codons V50 (GTT) or T50 (ACG), wild-type codon I50 (ATT) or polymorphic codon I50 (ATC).

Particularly preferred embodiments of the invention thus include a set of probes for codons 67-70 comprising at least one, preferably at least two, probe(s) for targetting at least one, preferably at least two, nucleotide changes in any of the following codons as represented in region III in FIG. 1:

wild-type K65 (AAA) or polymorphic K65 (AAG), wild-type K66 (AAA) or polymorphic K66 (AAG). wild-type D67 (GAC) or mutant N67 (AAC), wild-type T69 (ACT) or polymorphic T69 (ACA), mutant D69 (GAT) or N69 (AAT) or A69 (GCT), wild-type K70 (AAA), polymorphic K70 (AAG) or mutant R70 (AGA).

Particularly preferred embodiments of the present invention include a set of probes for codons 74-75 comprising at least one, preferably at least two, probes for targetting at least one, preferably at least two. nucleotide changes in any of the following codons as represented in region IV of FIG. 1:

wild-type K73 (AAA) or polymorphic K73 (AAG), wild-type L74 (TTA) or mutant V74 (GTA), wild-type V75 (GTA) or polymorphic V75 (GTG) or mutant T75 (ACA), wild-type D76 (GAT) or polymorphic D76 (GAC).

Particularly preferred embodiments of the present invention include a set of probes for codon 151 comprising at least one, preferably at least two, probes for targetting at least one, preferably at least two, nucleotide changes in any of the following codons as represented in region V of FIG. 1:

wild-type L149 (CTT) or polymorphic L149 (CTC) or L149 (CTG), wild-type P150 (CCA) or polymorphic P150 (CCG), wild-type Q151 (CAG) or mutant MI51 (ATG) or L151 (CTG) or polymorphic Q151 (CAA).

Particularly preferred embodiments of the present invention include a set of probes for codon 181-184 comprising at least one, preferably at least two, probe(s) for targetting at least one, preferably at least two, nucleotide changes in any of the following codons as represented in region VI of FIG. 1:

wild-type Y181 (TAT) or mutant C181 (TGT), wild-type Q182 (CAA) or polymorphic Q182 (CAG), wild-type Y183 (TAC) or polymorphic Y183 (TAT), wild-type M184 (ATG) or mutant V184 (GTG) or I184 (ATA) or G184 (AGG), wild-type D185 (GAT) or polymorphic D185 (GAC), wild-type D186 (GAT) or polymorphic E186 (GAG), wild-type L187 (TTA) or polymorphic G187 (GGA) or V187 (GTA).

Particularly preferred embodiments of the present invention include a set of probes for codon 215 comprising at least one, preferably at least two, probe(s) for targetting at least one, preferably at least two, nucleotide changes in any of the following codons as represented in region VII of FIG. 1

wild-type G213 (GGA) or polymorphic G213 (GGG), wild-type F214 (TTT) or polymorphic F214 (TTC) or L214 (CTT) or L214 (TTA), wild-type T215 (ACC) or polymorphic T215 (ACT), mutant Y215 (TAC) or F215 (TTC).

Particularly preferred embodiments of the present invention include a set of probes for codon 219 comprising at least one, preferably at least two, probe(s) for targetting at least one, preferably at least two, nucleotide changes in any of the following codons as represented in region VIII of FIG. 1

wild-type D218 (GAC) or polymorphic D218 (GAT), wild-type K219 (AAA) or polymorphic K219 (AAG) or mutant Q219 (CAA) or E219 (GAA), wild-type K220 (AAA) or polymorphic K220 (AAG).

Examples of probes of the invention are represented in Tables 3 and 4, and FIGS. 2 and 3. In Table 3, the probes withheld after selection are indicated using the letter “y”. These probes of the invention are designed for attaining optimal performance under the same hybridization conditions so that they can be used in sets of at least 2 probes for simultaneous hybridization; this highly increases the usefulness of these probes and results in a significant gain in time and labour. Evidently, when other hybridization conditions would be preferred, all probes should be adapted accordingly by adding or deleting a number of nucleotides at their extremities. It should be understood that these concommitant adaptations should give rise to essentially the same result, namely that the respective probes still hybridize specifically with the defined target. Such adaptations might also be necessary if the amplified material should be RNA in nature and not DNA as in the case for the NASBA (nucleic acid sequence-based amplification) system.

The selection of the preferred probes of the present invention is based on a reverse hybridization assay using immobilized oligonucleotide probes present at distinct locations on a solid support. More particularly the selection of preferred probes of the present invention is based on the use of the Line Probe Assay (LiPA) principle which is a reverse hybridization assay using oligonucleotide probes immobilized as parallel lines on a solid support strip (Stuyver et al. 1993; international application WO 94/12670). This approach is particularly advantageous since it is fast and simple to perform. The reverse hybridization format and more particularly the LiPA approach has many practical advantages as compared to other DNA techniques or hybridization formats, especially when the use of a combination of probes is preferable or unavoidable to obtain the relevant information sought.

It is to be understood, however, that any other type of hybridization assay or format using any of the selected probes as described further in the invention, is also covered by the present invention.

The reverse hybridization approach implies that the probes are immobilized to certain locations on a solid support and that the target DNA is labelled in order to enable the detection of the hybrids formed.

Methods for detecting nucleotide changes in RT genes of other viruses which have been found to harbour a pattern of drug-resistance mutation similar to the one observed for HIV based on the same principles as set out in the present invention should be understood as also being covered by the scope of the present invention.

The following definitions serve to illustrate the terms and expressions used in the present invention.

The term “antiviral drugs” refers particularly to an antiviral nucleoside analog or any other RT inhibitor. Examples of such antiviral drugs and the mutation they may cause in the HIV-RT gene are disclosed in Schinazi et al., 1994 and Mellors et al., 1995. The contents of the latter two documents particularly are to be considered as formina part of the present invention. The most important antiviral drugs focussed at in the present invention are disclosed in Tables 1 to 2.

The term “drug-induced mutation” refers to a mutation in the HIV RT gene which provokes a reduced susceptibility of the isolate to the respective drug.

The target material in the samples to be analysed may either be DNA or RNA, e.g.: genomic DNA, messenger RNA, viral RNA or amplified versions thereof. These molecules are also termed polynucleic acids.

It is possible to use genomic DNA or RNA molecules from HIV samples in the methods according to the present invention.

Well-known extraction and purification procedures are available for the isolation of RNA or DNA from a sample (f.i. in Maniatis et al., Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold Spring Harbour Laboratory Press (1989)).

The term “probe” refers to single stranded sequence-specific oligonucleotides which have a sequence which is complementary to the target sequence to be detected.

The term “target sequence” as referred to in the present invention describes the nucleotide sequence of the wildtype, polymorphic or drug induced variant sequence of the RT gene to be specifically detected by a probe according to the present invention. This nucleotide sequence may encompass one or several nucleotide changes. Target sequences may refer to single nucleotide positions, codon positions, nucleotides encoding amino acids or to sequences spanning any of the foregoing nucleotide positions. In the present invention said target sequence often includes one or two variable nucleotide positions. It is to be understood that the complement of said target sequence is also a suitable target sequence in some cases. The target sequences as defined in the present invention provide sequences which should be complementary to the central part of the probe which is designed to hybridize specifically to said target region.

The term “complementary” as used herein means that the sequence of the single stranded probe is exactly the (inverse) complement of the sequence of the single-stranded target, with the target being defined as the sequence where the mutation to be detected is located.

Since the current application requires the detection of single basepair mismatches, very stringent conditions for hybridization are required, allowing in principle only hybridization of exactly complementary sequences. However, variations are possible in the length of the probes (see below), and it should be noted that, since the central part of the probe is essential for its hybridization characteristics, possible deviations of the probe sequence versus the target sequence may be allowable towards head and tail of the probe, when longer probe sequences are used. These variations, which may be conceived from the common knowledge in the art, should however always be evaluated experimentally, in order to check if they result in equivalent hybridization characteristics than the exactly complementary probes.

Preferably, the probes of the invention are about 5 to 50 nucleotides long, more preferably from about 10 to 25 nucleotides. Particularly preferred lengths of probes include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. The nucleotides as used in the present invention may be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups which do not essentially alter their hybridisation characteristics.

Probe sequences are represented throughout the specification as single stranded DNA oligonucleotides from the 5′ to the 3′ end. It is obvious to the man skilled in the art that any of the below-specified probes can be used as such, or in their complementary form, or in their RNA form (wherein T is replaced by U).

The probes according to the invention can be prepared by cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequences, if need be by cleaving the latter out from the cloned plasmids upon using the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight. The probes according to the present invention can also be synthesized chemically, for instance by the conventional phospho-triester method.

The term “solid support” can refer to any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low. Usually the solid substrate will be a microtiter plate, a membrane (e.g. nylon or nitrocellulose) or a microsphere (bead) or a chip. Prior to application to the membrane or fixation it may be convenient to modify the nucleic acid probe in order to facilitate fixation or improve the hybridization efficiency. Such modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic groups, NH₂ groups, SH groups, carboxylic groups, or coupling with biotin, haptens or proteins.

The term “labelled” refers to the use of labelled nucleic acids. Labellina may be carried out by the use of labelled nucleotides incorporated during the polymerase step of the amplification such as illustrated by Saiki et al. (1988) or Bej et al. (1990) or labelled primers, or by any other method known to the person skilled in the art. The nature of the label may be isotopic (³²P, ³⁵S, etc.) or non-isotopic (biotin, digoxigenin, etc.).

The term “primer” refers to a single stranded oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products. Preferably the primer is about 5-50 nucleotides long. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strenght.

The fact that amplification primers do not have to match exactly with the corresponding template sequence to warrant proper amplification is amply documented in the literature (Kwok et al., 1990).

The amplification method used can be either polymerase chain reaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu & Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992) or amplification by means of Qβ replicase (Lizardi et al., 1988; Lomeli et al., 1989) or any other suitable method to amplify nucleic acid molecules known in the art.

The oligonucleotides used as primers or probes may also comprise nucleotide analogues such as phosphorothiates (Matsukura et al., 1987), alkylphosphorothiates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or may contain intercalating agents (Asseline et al., 1984).

As most other variations or modifications introduced into the original DNA sequences of the invention these variations will necessitate adaptions with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However the eventual results of hybridisation will be essentially the same as those obtained with the unmodified oligonucleotides.

The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, biological stability of the oligonucleotide molecules, etc.

The “sample” may be any biological material taken either directly from the infected human being (or animal), or after culturing (enrichment). Biological material may be e.g. expectorations of any kind, broncheolavages, blood, skin tissue, biopsies, sperm, lymphocyte blood culture material, colonies, liquid cultures, faecal samples, urine etc.

The sets of probes of the present invention will include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more probes. Said probes may be applied in two or more distinct and known positions on a solid substrate. Often it is preferable to apply two or more probes together in one and the same position of said solid support.

For designing probes with desired characteristics, the following useful guidelines known to the person skilled in the art can be applied.

Because the extent and specificity of hybridization reactions such as those described herein are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. The importance and effect of various assay conditions, explained further herein, are known to those skilled in the art.

The stability of the [probe: target] nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished by avoiding long AT-rich sequences. by terminating the hybrids with G:C base pairs, and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and % GC result in a Tm about 2-10° C. higher than the temperature at which the final assay will be performed. The base composition of the probe is significant because G-C base pairs exhibit greater thermal stability as compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving complementary nucleic acids of higher G-C content will be stable at higher temperatures.

Conditions such as ionic strenght and incubation temperature under which a probe will be used should also be taken into account when designing a probe. It is known that hybridization will increase as the ionic strenght of the reaction mixture increases, and that the thermal stability of the hybrids will increase with increasing ionic strenght. On the other hand, chemical reagents, such as formamide, urea, DMSO and alcohols, which disrupt hydrogen bonds, will increase the stringency of hybridization. Destabilization of the hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridization for synthetic oligonucleotide probes of about 10-50 bases in length occurs approximately 5° C. below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched base sequences to hybridize and can therefore result in reduced specificity.

It is desirable to have probes which hybridize only under conditions of high stringency. Under high stringency conditions only highly complementary nucleic acid hybrids will form: hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. The degree of stringency is chosen such as to maximize the difference in stability between the hybrid formed with the target and the nontarget nucleic acid. In the present case, single base pair changes need to be detected, which requires conditions of very high stringency.

The length of the target nucleic acid sequence and, accordingly, the length of the probe sequence can also be important. In some cases, there may be several sequences from a particular region, varying in location and length, which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another which differs merely by a single base. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly complementary base sequence will normally primarily determine hybrid stability. While oligonucleotide probes of different lengths and base composition may be used, preferred oligonucleotide probes of this invention are between about 5 to 50 (more particularely 10-25) bases in length and have a sufficient stretch in the sequence which is perfectly complementary to the target nucleic acid sequence.

Regions in the target DNA or RNA which are known to form strong internal structures inhibitory to hybridization are less preferred. Likewise, probes with extensive self-complementarity should be avoided. As explained above, hybridization is the association of two single strands of complementary nucleic acids to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. There can be intramolecular and intermolecular hybrids formed within the molecules of one type of probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization may be greatly increased. Computer programs are available to search for this type of interaction. However, in certain instances, it may not be possible to avoid this type of interaction.

Standard hybridization and wash conditions are disclosed in the Materials & Methods section of the Examples. Other conditions are for instance 3×SSC (Sodium Saline Citrate), 20% deionized FA (Formamide) at 50° C.

Other solutions (SSPE (Sodium saline phosphate EDTA), TMACl (Tetramethyl ammonium Chloride), etc.) and temperatures can also be used provided that the specificity and sensitivity of the probes is maintained. If need be, slight modifications of the probes in length or in sequence have to be carried out to maintain the specificity and sensitivity required under the given circumstances.

In a more preferential embodiment, the above-mentioned polynucleic acids from step (i) or (ii) are hybridized with at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen. fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more of the above-mentioned target region specific probes, preferably with 5 or 6 probes, which, taken together, cover the “mutation region” of the RT gene.

The term “mutation region” means the region in the HIV RT gene sequence where most of the mutations responsible for antiviral drug resistance or other observed polymorphisms are located. A preferred part of this mutation region is represented in FIG. 1. This mutation region can be divided into 8 important parts: drug induced variations and polymorphisms located within aa positions 38 to 44 of RT gene, drug induced variations and polymorphisms located within aa positions 47 to 53 of RT gene, drug induced variations and polymorphisms located within aa positions 65 to 72 of the RT gene, drug induced variations and polymorphisms located within aa positions 73 to 77 of the RT gene, drug-induced variations and polymorphisms located within aa positions 148 to 154 of the RT gene, drug-induced variations and polymorphisms located within aa positions 780 to 187 of the RT gene, drug induced variations and polymorphisms located within aa positions 212 to 216 of the RT gene and drug induced variations and polymorphisms located within aa positions 217 to 220 of the RT gene.

Since some mutations may be more frequently occurring than others, e.g. in certain geographic areas or in specific circumstances (e.g. rather closed communities) it may be appropiate to screen only for specific mutations, using a selected set of probes as indicated above. This would result in a more simple test, which would cover the needs under certain circumstances.

In order to detect the antiviral drug RT resistance pattern with the selected set of oligonucleotide probes, any hybridization method known in the art can be used (conventional dot-blot, Southern blot, sandwich, etc.).

However, in order to obtain fast and easy results if a multitude of probes are involved, a reverse hybridization format may be most convenient.

In a preferred embodiment the selected set of probes are immobilized to a solid support in known distinct locations (dots, lines or other figures). In another preferred embodiment the selected set of probes are immobilized to a membrane strip in a line fashion. Said probes may be immobilized individually or as mixtures to delineated locations on the solid support.

A specific and very user-friendly embodiment of the above-mentioned preferential method is the LiPA method, where the above-mentioned set of probes is immobilized in parallel lines on a membrane, as further described in the examples.

The invention also provides for any probes and primer sets designed to specifically detect or amplify specifically these RT gene polymorphisms, and any method or kits using said primer and probes sets.

The invention further provides for any of the probes as described above, as well as compositions comprising at least one of these probes.

The invention also provides for a set of primers allowing amplification of the mutation region of the RT gene in general.

Primers may be labeled with a label of choice (e.g. biotine). Different primer-based target amplification systems may be used, and preferably PCR-amplification, as set out in the examples. Single-round or nested PCR may be used.

The invention also provides for a kit for inferring the nucleotide sequence at codons of interest in the HIV RT gene and/or the amino acids corresponding to these codons and/or the antiviral drug resistance spectrum of HIV isolates present in a biological sample comprising the following components:

(i) when appropiate, a means for releasing, isolating or concentrating the polynucleic acids present in said sample;

(ii) when appropriate, at least one of the above-defined set of primers;

(iii) at least two of the probes as defined above, possibly fixed to a solid support;

(iv) a hybridization buffer, or components necessary for producing said buffer;

(v) a wash solution, or components necessary for producing said solution;

(vi) when appropriate, a means for detecting the hybrids resulting from the preceding hybridization.

(vii) when appropriate, a means for attaching said probe to a solid support.

The term “hybridization buffer” means a buffer enabling a hybridization reaction to occur between the probes and the polynucleic acids present in the sample, or the amplified products, under the appropiate stringency conditions.

The term “wash solution” means a solution enabling washing of the hybrids formed under the appropiate stringency conditions.

A line probe assay (LiPA) was designed for the screening for variations at interesting amino acids in the HIV RT gene. The principle of the assay is based on reverse hybridization of an amplified polynucleic acid fragment such as a biotinylated PCR fragment of the HIV RT gene onto short oligonucleotides. The latter hybrid can then, via a biotine-streptavidine coupling, be detected with a non-radioactive colour developing system.

The present invention further relates to a reverse hybridization method wherein said oligonucleotide probes are immobilized, preferably on a membrane strip.

The present invention also relates to a composition comprising any of the probes as defined in Tables 3 and 4 or FIGS. 2 and 3.

The present invention relates also to a kit for inferring the HIV RT resistance spectrum of HIV in a biological sample, coupled to the identification of the HIV isolate involved, comprising the following components:

(i) when appropiate, a means for releasing, isolating or concentrating the polynucleic acids present in the sample;

(ii) when appropriate, at least one of the sets of primers as defined above;

(iii) at least one of the probes as defined above, possibly fixed to a solid support;

(iv) a hybridization buffer, or components necessary for producing said buffer;

(v) a wash solution, or components necessary for producing said solution;

(vi) when appropriate, a means for detecting the hybrids resulting from the preceding hybridization;

(vii) when appropriate, a means for attaching said probe to a solid support

The following examples only serve to illustrate the present invention. These examples are in no way intended to limit the scope of the present invention.

Table 1: Mutations in HIV-1 RT gene associated with resistance against nucleoside RT inhibitors. More details are given in Mellors et al., 1995.

Table 2: Mutations in HIV-1 RT gene associated with resistance against HIV-1 specific RT inhibitors. For more details see Mellors-et al., 1995.

Abbreviations in Table 1 and 2:

AZT: 3′-azido-2′3′-dideoxythymidine

ddC: 2′3′-dideoxycytidine

ddl: 2′3′-dideoxyinosine

3TC: 3′dideoxy-3′-thiacytidine

FTC: 2′3′dideoxy-5-fluoro-3′-thiacytidine

L′697.593: 5-ethyl-6-methyl-3-(2-phthalimido-ethyl)pyridin-2(1H)-one

L′697.661: 3-Il(4,7-dichloro- 1,3-benzoxazol-2-yl)methyl amino-5-ethyl-6-methylpyridin-2(1H)-one Nevirapine: 1l-cyclopropyl-5,1]-dihydro-4-methyl-6H-dipyridol(3,2-b:2′,3′-e)diazepin-6-one

TIBO R82150: (+)-(5S)-4,5,6,7,-tetrahydro-5-methyl-6-(3-methyl-2butenyl)imidazo(4,5,1-)k) (1,4)-benzodiazepin-2(1H)-thione

TIBO 82913: (+)-(5S)-4,5,6,7, -tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)imidazo(4,5,1 kj)-(1,4)benzo-diazepin-2(1H)-thione TSAO-m²T: (2′,5′-bis-o-(tert-buthyldimethylsilyl)-3′-spiro-5′-(4′-amino-1′,2′-oxathiole-2′,2′-dioxide)

U90152: 1-(3-(1-methylethyl)-amino)-2-pyridinyl)-4-(5-(methylsulphonyl)-amino)-1H-indol-2yl)-carbonyl)-piperazine

Table 3: HIV RT wild-type and drug resistance mutation probes. The probes witheld after selection are indicated as “y”.

Table 4: Prediction and prevalence of LiPA probe reactivity. Probe names corresponding with the selected motifs are presented in the left column, with the relevant part of each probe shown under the consensus. The prevalence of these motives, determined using panels of European and US sera, is given in the middle column. The right column indicates the corresponding strips of FIGS. 2A-F and the accession number of the reference panel clone used to obtain this reactivity.

EXAMPLES Example 1

a. Materials and Methods.

Plasma samples were taken from HIV type-1 infected patients and stored at −20° C. until use. Patients were treated with AZT, ddI, ddC, D4T, 3TC, or several combinations of these prodrugs. The European serum samples tested were randomly selected. For the US serum collection, only the first sample from a follow-up series was taken. Some of these US patients were treated, others were not treated.

HIV RNA was prepared using the guanidinium-phenol procedure. Fifty μl plasma was mixed with 150 μl Trizol®LS Reagent (Life Technologies, Gent, Belgium) at room temperature (volume ratio: 1 unit sample/3 units Trizol). Lysis and denaturation occured by carefully pipetting up and down several times, followed by an incubation step at room temperature for at least 5 minutes. Fourty μl CHCl₃ was added and the mixture was shaken vigorously by hand for at least 15 seconds, and incubated for 15 minutes at room temperature. The samples were centrifuged at maximum 12,000 g for 15 minutes at 4° C., and the colourless aquous phase was collected and mixed with 100 μl isopropanol. To visualize the minute amounts of viral RNA, 20 μl of μg/μl Dextran T500 (Pharmacia) was added, mixed and left at room temperature for 10 minutes. Following centrifugation at max. 12,000 g for 10 minutes at 4° C. and aspiration of the supernatant, the RNA pellet was washed with 200 μl ethanol, mixed by vortexing and collected by centrifugation at 7,500 g for 5 minutes at 4° C. Finally the RNA pellet was briefly air-dryed and stored at −20° C.

For cDNA synthesis and PCR amplification, the RNA pellet was dissolved in 15 μl random primers (20 ng/μl, pdN₆, Pharmacia), prepared in DEPC-treated or HPLC grade water. After denaturation at 70° C. for 10 minutes, 5 μl cDNA mix was added, composed of 4 μl 5× AMV-RT buffer (250 mM Tris.HCl pH 8.5, 100 mM KCl, 30 mM MgCl₂, 25 mM DTT), 0.4 μL 25 mM dXTPs, 0.2 μl or 25U Ribonuclease Inhibitor (HPRI, Amersham), and 0.3 μl or 8 U AMV-RT (Stratagene). cDNA synthesis occured during the 90 minutes incubation at 42° C. The HIV RT gene was than amplified using the following reaction mixture: 5 μl cDNA, 4.5 μl 10× Taq buffer, 0.3 μl 25 mM dXTPs, 1 μl (10 pmol) of each PCR primer, 38 μl H₂O, and 0.2 μl (1 U) Taq. The primers for amplification had the following sequence: outer sense RT-9: 5′ bio-GTACAGTATTAGTAGGACCTACACCTGTC 3′ (SEQ ID NO 96); nested sense RT-1: 5′ bio-CCAAAAGTTAAACAATGGCCATTGACAGA 3′ (SEQ ID NO 97); nested antisense RT4: 5′ bio-AGTTCATAACCCATCCAAAG 3′ (SEQ ID NO 98); and outer antisense primer RT-12: 5′ bio-ATCAGGATGGAGTTCATAACCCATCCA 3′ (SEQ ID NO 99). Annealing occured at 57° C., extension at 72° C. and denaturation at 94° C. Each step of the cycle took 1 minute, the outer PCR contained 40 cycles, the nested round 35. Nested round PCR products were analysed on agarose gel and only clearly visible amplification products were used in the LiPA procedure. Quantification of viral RNA was obtained with the HIV Monitor™test (Roche, Brussels, Belgium).

Selected PCR products, amplified without 5′ biotine primers, were cloned into the pretreated EcoRV site of the pGEMT vector (Promega). Recombinant clones were selected after α-complementation and restriction fragment length analysis, and sequenced with plasmid primers and internal HIV RT primers. Other biotinylated fragments were directly sequenced with a dye-terminator protocol (Applied Biosystems) using the amplification primers. Alternatively, nested PCR was carried out with analogs of the RT-4 and RT-1 primers, in which the biotine group was replaced with the T7- and SP6-primer sequence, respectively. These amplicons were than sequenced with an SP6- and T7-dye-primer procedure. Sequence information was submitted to the GENBANK.

Probes were designed to cover the different polymorphisms and drug induced mutations. In principle, only probes that discriminated between one single nucleotide variation were retained. However, for certain polymorphisms at the extreme ends of the the probe, cross-reactivity was tolerated. Specificity was reached for each probe individually after considering the % (G+C), the probe lenght, the final concentration of the buffer components, and hybridization temperature. Optimized probes were provided enzymatically with a poly-T-tail using the TdT (Pharmacia) in a standard reaction condition, and purified via precipitation. Probe pellets were disolved in standard saline citrate (SSC) buffer and applied as horizontal parallel lines on a membrane strip. Control lines for amplification (probe 5′ CCACAGGGATGGAAAG 3′, HIV RT aa 150 to aa 155) and conjugate incubation (biotinylated DNA) were applied alongside. After fixation of the probes onto the membranes by baking, membranes were sliced into 4 mm strips.

To perform LiPA tests, equal amounts (10 μl) of biotinylated amplification products and denaturation mixture (0.4 N NaOH/0.1% SDS) were mixed, followed by an incubation at room temperature for 5 minutes. Following this denaturation step, 2 ml hybridization buffer (2×SSC, 0.1% SDS, 50 mM Tris pH7.5) was added together with a membrane strip and hybridization was carried out at 39° C. for 30 min. Then, the hybridization mixture was replaced by stringent washing buffer (same composition as hybridisation buffer), and stringent washing occured first at room temperature for 5 minutes and than at 39° C. for another 25 minutes. Buffers were than replaced to be suitable for the streptavidine alkaline phosphatase conjugate incubations. After 30 minutes incubation at room temperature, conjugate was rinsed away and replaced by the substrate components for alkaline phosphatase, Nitro-Blue-Tetrazolium and 5-Bromo4-Chloro-3-Indolyl Phosphate. After 30 minutes incubation at room temperature, probes where hybridization occured became visible because of the purple brown precipitate at these positions.

b. Results.

b.1 The HIV-1 RT gene PCR and selection of a reference panel.

PCR primers were chosen outside the target regions for probe design. The amplified region located inside the nested primers covered the HIV-1 RT gene from codon 29 to codon 220. The primer design was based on published sequences from the HIV-1 genotype B lade. European and United States HIV-1 positive serum samples, stored appropriately (at −20° C.) without repeating freezing-thawing cycles, were PCR positive in 96% of the cases (not shown). The annealing temperature for the selected primers seemed to be crucial (57° C.). At 55° C., a second aspecific amplicon of approximately 1500-bp was generated; and at 59° C. the amount of specific fragment decreased drastically. With the current primer combination, the corresponding RT region could be amplified from isolates of the genotype A, C, D and F lade, but with a reduced sensitivity.

A total of 25 selected PCR fragments with the target polymorphisms and mutations were retained as reference panel and sequenced on both strands. The selection occurred during the evaluation of the probes, and these samples originated from naive or drug-treated European or US patients. Biotinylated PCR products from this panel (Accession Number L78133 to L78157) were used to test probes for specificity and sensitivity.

b.2 Nucleotide target region for probe design and probe selection.

Table 4 and parts of FIG. 1 are a compilation of the natural and drug-selected variability in the vicinity of aa 41, 69-70, 74-75, 184, 215, and 219 of the HIV RT gene.

To create this table and parts of this figure, the “National Centre for Biotechnology Information” database was searched and all HIV-1 genome entries were retrieved and analyzed one by one. Only those entries displaying non-ambiguous sequence information in the vicinity of the above-mentioned codons were retained for further interpretation. It should be noted that the indicated variations do not imply that they occur in the same sequence: for example the variability observed at codon 40 and 43 may occasionally occur together, but most often, if they occur, only one of them is found. In these 6 regions, a total of 19 different third-letter and two first-letter (codon 43 AAG versus GAG and codon 214 TTT versus CTT) polymorphisms need to be included in the selection of wild type probes. Another 13 first-letter and/or second-letter variations are drug-induced and are the main targets for the selection of probes (FIG. 1).

For the design of relevant probes, only those database motifs that systematically returned (highly prevalent motif) were included, while scattered mutations which were found randomly (low prevalent motif, not shown) were ignored. Based on database sequences seven motifs for codon 41 (91.6% of all entrees), 6 for codon 69-70 (86.2%), 2 for codon 74-75 (90.4%), 5 for codon 184 (96.6%), 9 for codon 215 (94.1%), and 2 for codon 219 (88.2%) were selected (Table 4).

Probe names corresponding with the selected motifs are presented in the left colunr of Table 4, with the relevant sequence part of each probe shown under the consensus. The prevalence of these motives was than determined using panels of European and US sera (Table 4).

In many cases, the database entries were not representative for the samples tested. Upon analyzing the European and US samples, many were not reactive with these database-selected probes. Upon sequencing analysis of several of these unreactive PCR products, another 8 motifs became apparent, for which the corresponding probes were designed (41w20, 41m12, 70m13, 74w9, 74m6, 74 m12, 184w24, 215 m49). By including these newly designed motifs, negative results were markedly decreased; for all codon positions except codon 219, the total percentage of reactivity exceeds 90%.

Another 4 probes were designed (41m11, 215m50, 219m7, and 219m9) because their sequence motif was found in the cloned reference panel, although no reactivity with the tested plasma virus samples was found so far. The existence of these rare sequence motifs is explained by assuming that they exist at an extremely low frequency in the viral quasispecies, remaining undetectable by direct detection methods, but becoming apparent after cloning.

The sequence motif of probe 215m13 was Generated in recombinant clones by site-directed mutagenesis (not shown). The rational behind this probe design was to determine whether the sequence combination of codon Y215 (TAC) can occur in combination with L214 (CTT) in vivo. However, this latter motif was not found in the plasma samples tested.

Four probes (41w15, 70w8, 215w29, 215w27) are in fact redundant, because they detect identical sequence motifs covered by other probes. However. the location of these redundant probes is slightly different to their sequence-identical counterpart. These probes have the potential to avoid negative results which might otherwise appear as a consequence of random mutations in the probe target area and can therfore increase the specificity of recognition.

b.3 Probe specificity and sensitivity.

The 48 selected probes were applied separately on LiPA strips. Biotinylated PCR fragments generated from the reference panel or directly from plasma virus were alkali-denatured, the hybridization buffer and LiPA strips were added, and submitted to stringent hybridization and washing conditions. Positions where hybridization occurred were revealed by the biotine-streptavidine colorimetric detection system. FIG. 2 (A to F) shows the reactivity of these 48 designed probes. In the right columns of Table 4, there is the indication of the corresponding strip in FIG. 2, and the accession number of the reference panel clone used to obtain this reactivity. The reactivities of these probes were concordant with the nucleotide sequences. False positive reactivities were observed only for probe 41w19 (FIG. 2A.9) and for 70m3 (FIG. 2B.8), with extremely rare sequence motifs 41m12 (prevalence less than 0.3%) and 70m16 (not experimentally found), respectively. Weak cross-reactivity, as was observed on probe 41m13 with a 41m27 motif (FIG. 2A.10) was, in general, not tolerated in the probe design. When occurring, however, it never influenced the genotypic resistance interpretation.

b.4 Applicability of the LiPA in patient management.

We selected follow-up samples from three patients and analyzed the viral genotype on the 48 LiPA probes. FIG. 3 illustrates the applicability of genotypic resistance measurement in conjunction with the analysis of viral load and CD4 cell count. All three patients had a wild type virus (i.e. M41-T69-K70-L74-V75-M184-F214-T219-K219) strain in the sample collected before anti-retroviral treatment. Codon positions that changed upon treatment are presented in FIG. 3.

From Patient 91007, 11 serum samples were analyzed, the first sample being collected 2 weeks before the start of therapy. The LiPA revealed that before treatment, in a T215 context, two variants at codon position 213 were predominantly present (GGG and GGA respectively detected by probe 215w11 and 215w9/215w29). From week 50 until week 81, a mixture of T215 and Y215 could be detected. Both variants at codon 213 were also represented in the selected resistant genotypes (probes 215m17 and 215m14 are positive). From week 94 onwards, only Y215 mutant virus could be detected. A nearly identical geno-conversion at codon 41 was observed, with the detection of mixtures (M41 and L41) from week 81 until week 111; from week 126 onwards, only L41 could be found (strips not shown). CD4 values were highly variable. Nevertheless, a continuous decrease in CD4 is apparent (p=0.019, linear regression analysis). Viral load also decreased initially. However, the direct response to the treatment might have been missed in this follow-up series, since the first sample after the start of the treatment is at 32 weeks. From than on, viral load increased.

Patient 94013 was treated with 3TC monotherapy from week 2 onwards. At week 10, a mixture of M184 and V184 could be detected. From week 14 on, only V184 was present. CD4 counts increased nearly 2.5-fold, with the highest level at week 10. Viral load decreased spectacularly by 3 log units. But from week 10 onwards, a slight but steady increase to week 23 was noted. The decrease in CD4 and increase in viral load coincided with the appearance of the V184 motif.

Patient 92021 was followed for 55 weeks. AZT treatment started at week 10, followed by a supplemental ddC treatment from week 20 onwards. The first sample was found to be reactive with probe 215w9/w29 (F214T215=TTTACC), but trace amounts of reactivity with 215w53 (L214T215=TTAACC) could be detected as well, indicating the presence of at least two variants at that time. From week 19 onwards, the codon L214=TTA motif became more important. At week 42, the first sign of genotypic resistance could be detected by the presence of a F214Y215 motif (TTTTAT). Finally at week 55, only F214Y215 could be detected. The L214 TTA motif disappeared completely. At week 42. a mixture (K and R) at codon 70 was present, but at week 55, only R70 could be detected. At week 55, a mixture of codon 219 motifs (K and E) was found (strips not shown). CD4 initially increased, with a maximal effect during AZT monotherapy peaking at week 21. From then on, a continuous decrease was observed. However, ten weeks of AZT treament did not result in a drop in viral load, since the values of week 16 and 19 were nearly unchanged. It is only after start of the combination therapy (week 20) that the viral load dropped by 1.67 log. From this patient, it is tempting to assume that L214T215 confers genotypic resistance to AZT treatment, and that the addition of ddC is necessary to induce the natural F214Y215 genotype. The rise in CD4 cell count may be the consequence of the drug itself, and not from drug-induced protection (Levy et al. 1996).

c. Discussion

By adapting the previously designed LiPA technology (Stuyver et al. 1993) for the HIV RT gene, the described assay format permits the rapid and simultaneous detection of wild type and drug-selected variants associated with the genotypic resistance for AZT, ddI, ddC, d4T. FTC and 3TC. The Inno LiPA HIV drug-resistance strip provides information about the genetic constitution of the RT gene in the vicinity of codon 41, 69, 70, 74, 75, 184, 215, and 219 at the nucleotide and, hence, also at the deduced protein level. Essentially, the biotinylated RT PCR product is hybridized against immobilized specific oligonucleotides (Table 4), which are directed against the indicated codon variabilities. Following this reverse-hybridization, the oligonucleotide-biotinylated-PCR-strand hybrid is recognized by the streptavidine-alkaline phosphate conjugate, which then in turn converts the alkaline phosphate substrate into a purple brown precipitate.

Using this assay, we studied the specificity and reactivity of 48 probes, covering 6 different regions. This combination should allow the reliable detection of most of the genetic resistance-related codon combinations observed to date. Occasionally occurring mutations in the vicinity of the target codons, not taken into consideration during probe design, may eventually prevent hybridization of the probes for a particular target region. This problem is partially solved by the redundancy of probes at the most important codons. Results obtained using 358 HIV infected plasma samples showed that, depending on the codon position under investigation, between 82.4% and 100% of the combinations could be detected, or an average of 92.7%. It is important to mention here that the assay was developed for resistence detection of the HIV-1 genotype B, and only limited information is currently available about the outcome of this assay with other genotypes. Since the amplification primer combination is more or less universal for all the HIV-1 isolates, some of the indeterminate results may well be due to the presence of non-genotype B virus strains.

So far, several assays for the detection of the wild-type and drug-selected mutations in the HIV RT gene have been described. These include Southern blotting (Richman et al., 1991), primer-specific PCR (Larder et al., 1991), PCR-LDR (Frenkel et al., 1995), RNAse A mismatch cleaving (Galandez-Lopez et al., 1991), and hybridization against enzyme-labeled probes (Eastman et al., 1995). The general advantage of the LiPA and other genotypic assays is the speed by which results are obtained when compared to phenotypic assays. The particular advantage of our test is its multi-parameter (in this particular case multi-codon) format. Moreover, the assay can easily be extended not only for the screening of the other RT-codons, but also for proteinase codons associated with resistance (Mellors et al., 1995). As was illustrated in FIG. 3, mixtures of wild-type and drug-selected mutations can be detected easily. The detection limit for these mixtures is dependent on the sensitivity of the probes, but reliable results can be obtained as soon as 5 to 10% of the minor component is present (not shown). We were unable to provide reliable evidence for mixtures with any sequencing protocol at the same sensitivity level.

Due to the large amount of variables that need to be included in the selection of specific probes (temperature of hybridization, ionic strength of hybridization buffer, length of the probe, G+C content, strand polarity), it might occasionally occur that some of the probes will show weak false positive reaction with related but hitherto unreported sequences. In our experience, and if this occurred, this has never influenced the interpretation at the deduced aa level. In the current selection of probes, all except two (41w19 and 70m3) were retained on the basis of 100% specificity: as soon as one nucleotide differs in the probe area, hybridization is abolished. Further fine-tuning of these two probes will therefore be necessary to obtain the required specificity.

Accompanying polymorphisms in the vicinity of the target codons are found with a rather high prevalence in wild-type virus strains, but not in mutant sequences. A partial list of such combinations is hereby presented: codon V74=GTA without polymorphism at codon 73, 75 or 76; codon V184=GTG without codon Q182=CAG; and codon F215=TTC without F214=TTC/TTA or L214=CTT. The most intriguing example is the following: L214T215 (CTTACC) is predicted for approximately 7.8% of the wild type sequences. The corresponding motif L214Y215 (CTTTAT) apparently does not exist in plasma virus. From the example shown in FIG. 3, it is clear that selection of mutants is a very flexible and complex phenomena. In this particular case, viruses having codon F214 were replaced by a L214 viral population in the AZT monotherapy period, but upon selecting for genotypic drug resistance at codon 215, the original F214 configuration was restored. Clearly, the selection for the Y215 genotype prohibits the presence of a L214 genotype. Since no evidence has yet emerged that L214 confers resistance to anti-retroviral compounds, the appearance of this special mutant during the AZT monotherapy period is difficult to interpret. More research will certainly be necessary to clarify this issue. But if L214 should indeed provide low-level genotypic resistance to AZT treatment, approximately 7.8% of the naive infections will not benefit from initial AZT monotherapy.

Since antiviral treatment can result in a marked extension of life expectancy for HIV infected patients, it is of utmost importance to find the best drug regimen for each individual separately. Therefore, monitoring of the magnitude and duration of the virus load and CD4 cell changes is a prerequisite. However, knowledge concerning the genetic constitution of the virus may also be an important factor in designing optimal treatment schedules. Optimizing therapies making good use of available information (viral load, CD4 cell count, genetic resistance) has remained largely unexploited. If this was partially due to the complexity of screening for all the mutational events, the above-described LiPA technology should remove one key obstacle.

In conclusion, we have described a genotypic assay for the detection of wild type and drug selected codons in the HIV RT gene. The combination of the assay result along with viral load and CD4 cell monitoring should permit better design of patient-dependent optimal treatment schedules.

Example 2

Multi-Drug Resistant (MDR) HIV-1 isolates have been described. These MDR isolates are characterized by having mutations in their genome, compared to the wild type HIV-1 genome, which result in a set of amino acid changes. A key mutation leading to multi-drug resistance was found to be localized in codon 151 of the HIV-1 RT gene. Consequently, and as detecting these MDR isolates is clinically important, we designed probes recognizing wild-type (probe 151w2) and mutant (probes 151m4 and 151m19) HIV-1 isolates. Furthermore, the presence of polymorphisms in the direct vicinity of codon 151 (codon 149) and at codon 151 have been described. Therefore, we also designed two additional probes (probes 151w6 and 151w11) which detect these polymorphisms (FIG. 4 and Table 3).

Treatment with non-nucleoside analogues, such as Nevirapine (Boehringer Ingelheim), selects for several amino acid changes in conserved regions of the HIV-1 RT gene. One of the most important amino acid changes is Y181C, a codon change that confers high level resistance. As the detection of this mutation is also clinically important, we designed probes recognizing the wild-type (181w3 and 181w5) and mutant (181m7) isolates (FIG. 4 and Table 3).

FIG. 4 shows the application of the selected probes for codon 151 and 181. The position of the probes on the strips is indicated on the right side of the strips. LiPA strips were incubated with sequence-confirmed PCR fragments, extracted and amplified from: a wild type HIV-1 isolate (strip 1), a wild type HIV-1 isolate with a polymorphism at codon 151 (strip 2) or codon 149 (strip 3), a multi-drug resistant HIV-1 isolate (strip 4) with no information about codon 181 and a non-nucleoside analogue-treated HIV-1 isolate which remained wild type at codon 151(strip 5).

TABLE 1 AZT M41L ATG to TTG or CTG D67N GAC to AAC K70R AAA to AGA T215Y ACC to TAC T215F ACC to TTC K219Q AAA to CAA K219E AAA to GAA ddl K65R AAA to AGA L74V TTA to GTA V75T GTA to ACA M184V ATG to GTG ddC K65R AAA to AGA T69D ACT to GAT L74V TTA to GTA V75T GTA to ACA M184V ATG to GTG Y215C TTC to TGC d4T I50T ATT to ACT V75T GTA to ACA 3TC or M184V ATG to GTG or GTA FTC M184I ATG to ATA 1592U89 K65R AAA to AGA L74V TTA to GTA Y115F TAT to TTT M184V ATG to GTG

TABLE 2 Nevirapine A98G GCA to GGA L100I TTA to ATA K103N AAA to AAC V106A GTA to GCA V108I GTA to ATA Y181C TAT to TGT Y181I TGT to ATT Y188C TAT to TGT G190A GGA to GCA TIBO L199I TTA to ATA R82150 TIBO L100I TTA to ATA R82913 K103N AAA to AAC V106A GTA to GCA E138K GAG to AAG Y181C TAT to TGT Y188H TAT to CAT Y188L TAT to TTA L697,593 K103N AAA to AAC Y181C TAT to TGT L697,661 A98G GCA to GGA L100I TTA to ATA L697,661 K101E AAA to GAA K103N AAA to AAC K103Q AAA to CAA V108I GTA to GCA V179D GTT to GAT V179E GTT to GAG Y181C TAT to TGT BHAP U-90152 P236L CCT to CTT BHAP K101E AAA to GAA U-87201 K103N AAA to AAC Y181C TAT to TGT Y188H TAT to CAT E233V GAA to GTA P236L CCT to CTT K238T AAA to ACA BHAP L100I TTA to ATA U-88204 V106A GTA to GCA Y181C TAT to TGT Y181I TGT to ATT HEPT Y188C TAT to TGT E-EBU Y181C TAT to TGT E-EBU-dM Y106A GTA to GCA E-EPU and Y181C TAT to TGT E-EPSeU Y188C TAT to TGT a-APA Y181C TAT to TGT R18893 S-2720 G190E GGA to GAA TSAO E138K GAG to AAG BM +51.0836 Y181C TAT to TGT

TABLE 3 HIV RT wild-type and drug resistance SEQ ID NO PROBE Formula probe Sequentie oligo selection wild-type probes for position M41 E40M41K43 41w7 AGAAATGGAAAAGGA  1 y E40M41K43 41w15 TGTACAGAAATGGAA  2 y M41K43 41w16 AAATGGAAAAGGAAG  3 E40M41 41w18 TACAGAGATGGAAA  4 E40M41K43 41w19 GTACAGAGATGGAAA  5 E40M41K43 41w20 AGAGATGGAAAAAGA  6 y E40M41K43 41w30 AGAAATGGAGAAGGA  7 y E40M41 41w31 ACAGAGATGGAAAA  8 E40M41 41w32 GTACAGAGATGGAA  9 y E40M41K43 41w33 CAGAGATGGAAAAG  10 E40M41K43 41w34 AGAAATGGAAAAAGA  11 E40M41K43 41w35 GAAATGGAAAAAGA  12 E40M41K43 41w36 CAGAAATGGAAAAAGA  13 y E40M41K43 41w37 AGAAATGGAAAAAGAA  14 drug-induced variant probes for position L41 E40L41K43 41m8 AGAATTGGAAAAGGA  15 E40L41K43 41m11 AGAGTTGGAAAAGGA  16 y E40L41K43 41m12 AGAGCTGGAAAAGG  17 y E40L41K43 41m13 AGAACTGGAAAAGG  18 y E40L41K43 41m14 GAGCTGGAAAAGG  19 E40L41K43 41m21 ACAGAATTGGAAAAG  20 y E40L41 41m22 ACAGAATTGGAAAA  21 E40L41 41m23 ACAGAACTGGAAAA  22 E40L41K43 41m24 AGAATTGGAAGAGG  23 y E40L41E43 41m25 CAGAATTGGAAGAGG  24 E40L41E43 41m26 AGAATTGGAAGAGGA  25 E40L41E43 41m27 AGAACTGGAAGAGG  26 y E40L41E43 41m28 CAGAACTGGAAGAGG  27 E40L41E43 41m29 AGAACTGGAAGAGGA  25 wild-type probes for positions I50 or V50 or T50 K49I50 50w4 CAAAAATTGGGCCT  29 y R49I50 50w9 ATTTCAAGAATTGGG  30 y K49V50 50w5 TTCAAAAGTTGGGC  31 y K49I50 50w13 CAAAAATCGGGCCTG  32 y K49T50 50w14 AAAAATCGGGCCTGA  33 y wild-type probe for position D67 K64K65K66D67 67w4 AAAGAAGAAAGACAG  34 y drug-induced variant probe for position N67 K64K65K66N67 67m19 ATAAAGAAAAAGAACAGTA  35 y wild-type probes for positions T69 or K70 T69K70 70w1 AGTACTAAATGGAGAA  36 y D69K70 70w2 AGTGATAAATGGAGAA  37 y T69K70 70w5 ACAGTACTAAATGGAG  35 y K70K73 70w11 TAAATGGAGAAAAITAG  40 drug-induced variant probes for positions D69 or N69 or A69 or R70 D69R70 70m3 GTGATAGATGGAGAA  41 T69R70 70m6 GTACTAGATGGAGA  42 T69R70 70m12 AGTACTAGATGGAGA  43 y T69R70 70m13 AGTACAAGATGGAGA  44 y N69R70 70m14 CAGTAATAGATGGAG  45 y A69R70 70m15 ACAGTGCTAGATGGA  46 A69R70 70m16 CAGTGCTAGATGGA  47 y A69R70 70m17 CAGTGCTAGATGGA  46 D69R70 70m18 CAGTGATAGATGGA  49 y D69R70 70m19 CAGTGATAGATGGAG  50 D69R70 70m20 AGTGATAGATGGAG  51 D69R70 70m21 AGTGATAGATGGAGA  52 wild-type probes for positions L74 or V75 K73L74V75D76 74w5 GAGAAAATTAGTAGATTT  53 y K73L74V75D76 74w8 AAAATTAGTAGACTTC  54 y K73L74V75D76 74w9 GAGAAAGTTAGTGGATT  55 drug-induced variant probes for positions V74 or T75 K73V74V75D76 74m6 AGAAAAGTAGTAGATTT  56 y K73L74T75D76 74m10 AAAATTAACAGATTTC  57 K73L74T75D76 74m11 GAAAATTAACAGATTT  58 K73L74T75D76 74m12 GAAAATTAACAGATTTC  59 y wild-type probes for position Q151 Pl50Q151G152 151w2 CTTCCACAGGGATGG  60 y P150Q151G152 151w6 CTTCCACAAGGATGG  61 y P150Q151G152 151w11 TGCTCCCACAGGGATG  62 y drug-induced variant probe for position M151 P150M151G152 151m4 CTTCCAATGGGATGG  63 y P150M151Gl52 151m19 GCTTCCAATGGGATGG  64 y wild-type probe for position Y181 Y181 181w3 AGTTATCTATCAATACAG  65 y drug-induced variant probe for position C181 C181 181m7 AGTTATCTGTCAATAC  66 y wild-type probes for position M184 Q182M184 184w11 TCAATACATGGATGAGG  67 y Q182M184 184w17 TCAGTACATGGATGAGG  68 y Q182M184 184w18 ATCAATACATGGATGA  69 Q182M184 184w19 TCAGTACATGGATG  70 Q182M184 184w21 ATCAATATATGGATG  71 y Q182M184 184w22 ATCAATATATGGATGA  72 Q182M184 184w23 TCAATATATGGATGA  73 Q182M184 184w24 TCAATACATGGACGA  74 y Q182M184 184w25 CAATACATGGACGAT  75 Q182M184 184w26 TCAATACATGGACGAT  76 drug-induced variant probes for position V184 or I184 Q182V184 184m12 CAATACGTGGATGAGGG  77 y I184 184m13 AATACATAGATGAT  78 Q182I184 184m14 CAATACATAGATGAT  79 Q182I184 184m15 CAATACATAGATGATT  80 Q182V184 184m16 CAATACGTAGATGAT  81 Q182V184 184m20 TCAATACGTGGATGA  82 Q182I184 184m27 TCAATACATAGATGAT  83 Q182I184 184m28 ATCAATACATAGATGAT  84 y wild-type probes for position T215 G213F214T215 215w9 GGATTTACCACACCA  85 y L214T215 215w10 GACTTACCACACCA  86 y F214T215 215w11 GGTTTACCACACCA  87 y F214T215 215w16 GATTTACCACACCA  88 T215 215w22 TTACTACACCAGAC  89 y T215 215w24 TTACCACACCAGA  90 G213L214T215 215w27 TGGGGACTTACCAC  91 y G213F214T215 215w29 TGGGGATTTACCAC  92 y G213F214T215 215w32 GGGGTTCACCACAC  93 G213F214T215 215w33 GGGATTCACCACAC  94 y G213F214T215 215w34 GGGATTTACCACACCAG  95 G213L214T215 215w35 TGGGGACTTACCACACC  96 G213F214T215 215w36 TGGGGGTTTACCACACC  97 G213F214T215 215w37 GGGATTTACTACACCAG  98 G213L214T215 215w52 GGGATTAACCACAC  99 G213L214T215 215w53 GGGGATTAACCACA 100 y G213L214T215 215w54 TGGGGATTAACCACA 101 G213L214T215 215w55 GGGGGTTAACCACA 102 G213L214T215 215w56 GGGGTTAACCACAC 103 G213L214T215 215w57 TGGGGGTTAACCAC 104 G213L214T215 215w65 GGGATTGACCACAC 105 G213L214T215 215w66 GGATTGACCACACC 106 G213L214T215 215w67 GGGATTGACCACA 107 y G213L214T215 215w68 GGGACTGACCACA 108 y G213L214T215 215w69 GGGACTGACCACAC 109 G213L214T215 215w70 TGGGGGTTAACCACA 110 G213L214T215 215w71 TGTGGTTAACCCCCA 111 y G213L214T215 215w51 GGGGCTTACCACAC 112 drug-induced variant probes for position Y215 or F215 G213L214Y215 215m13 GGACTTTACACACC 113 y G213F214Y215 215m14 GGGTTTTACACACC 114 y G213F214F215 215m15 GGATTTTTCACACCA 115 G213F214Y215 215m17 GGATTTTACACACC 116 y G213F214Y215 215m38 GGGATTTTACACACCAG 117 G213F214F215 215m39 GGGATTTTTCACACCAG 118 G213F214Y215 215m40 GGGATTTTACACAC 119 G213F214Y215 215m41 GGGGATTTTACACA 120 G213F214Y215 215m43 CCCTAAAATGTGTG 121 G213F214F215 215m44 GGATTTTTCACACC 122 F214F215 215m45 GATTTTTCACACCA 123 y G213F214F215 215m46 GGGATTTTTCACAC 124 G213F214Y215 215m42 CCCCTAAAATGTGT 125 F214Y215 215m47 GGTTTTATACACCA 126 G213F214Y215 215m43 GGGTTTTATACACC 127 G213F214Y215 215m49 GGGGTTTTATACAC 128 y G213F214T215 215m50 GGGGGCTTACCACA 129 y G213F214Y215 215m61 GGATTCTACACACC 130 y F214Y215 215m62 GATTCTACACACC 131 G213F214Y215 215m63 GGATTCTACACAC 132 G213F214Y215 215m64 GGGATTCTACACAC 133 G213F214Y215 215m72 GGGTTTTATACCCC 134 F214Y215 215m73 GGTTTTATACCCC 135 F214Y215 215m74 GTTTTATACCCCA 136 wild-type probes for position K219 K219 219W1 ACCAGACAAAACA 137 K219 219w2 ACCAGACAAAAAAC 138 y K219 2l9w3 CACCAGACAAAAAAC 139 K219 219W13 CAGACAAGAAACAT 140 K219 219w14 CCAGACAAGAAACA 141 K219 219w15 ACCAGACAAGAAACA 142 K219 219w16 AGACAAAAAGCATC 143 y K219 2l9w17 CAGACAAAAAGCAT 144 K219 219w18 CAGACAAAAAGCATC 145 K219 2l9w19 CCAGATAAAAAACA 145 K219 219w20 ACCAGATAAAAAAC 147 K219 219w21 CCCAGATAAAAAACA 145 K219 219w22 CCAGATAAAAAACATC 149 K219 219w23 CACCAGATAAAAAAC 150 K219 219w24 CAGACAAGAAACATC 151 K219 219w25 ACCAGACAAGAAAC 152 drug-induced variant probes for position Q219 or E219 Q219 219m4 ACCAGACCAAAAACA 153 E219 219m5 ACCAGACGAAAAACA 154 Q219 219m6 ACCAGATCAAAAACA 155 Q219 219m7 ACCAGATCAAAAAC 156 y Q219 219m8 CACCAGATCAAAAAC 157 E219 219m9 ACCAGACGAAAAAC 155 y E219 219m10 CCAGACGAAAAACA 159 Q219 219m11 CCAGACCAAAAACA 160 Q219 219m12 ACCAGACCAAAAAC 161

TABLE 4 Prediction and prevalence of LiPA probe reactivity consensus prevalence Corresponding        nucleic acid database Europe  US  Rp Figure probe     Codon 38-43 amino acid n = 191/m = 25 n = 306 n = 52 n = 25 strip Acc. No TGTACAGAAATGGAAAAG CTEMEK 41w7       ------------   ---- 122 (62.9%) 237  35  11 1a.1 L78149 41w15* --------------- ----- 118 230  38   9 1a.1 L78149 41w19    -----G---  ----   5 (2.6%)  10   2   2 1a.2 L78156 41w20       --G--------A   ----   0   6   0   1 1a.3 L78157 41w30       --------G---   ----   1 (0.5%)   8   6   1 1a.4 L78154 41m21    ------T--------  --L--  18 (9.4%)  37   7   2 1a.5 L78136 41m11       --GT--------   -L--   0   0   0   1 1a.6 L78140 41m24       ---T-----G--   -L-E  12 (6.3%)   1   2   1 1a.7 L78144 41m13       ---C--------   -L--  14 (7.3%)  21   3   1 1a.8 L78139 41m12       --GC--------   -L--   0   1   0   1 1a.9 L78155 41m27       ---C-----G--   -L-E   3 (1.6%)   0   1   1 1a.10 L78137 total 175 (91.6%)  95.1% 100%  88% consensus prevalence Corresponding        nucleic acid database Europe  US  Rp Figure probe     Codon 68-72 amino acid n = 191/m = 25 n = 306 n = 52 n = 25 strip Acc. No AGTACTAAATGGAGA STKWR 70w1 --------------- ----- 224 (63.3%) 230  39  13 1b.1,2 L78147 70w8* ------------ ---- 208  210  38  11 1b.2 L78144 70m12 -------G------- --R--  37 (10.5%)  46   6   4 1b.3 L78148 70m13 -----A-G------- --R--   0   0   1   2 1b.4 L78133 70w2 ---GA---------- -D---  25 (7.1%)   4   4   2 1b.5 L78136 70m3     GA--G-------  DR--  10 (2.8%)   3   1   0 1b.6 pending 70m14 ----A--G---- -NR-   7 (2.0%)   4   5   2 1b.7 L78154 70m16 ---G---G---- -AR-   2 (0.6%)   0   0   1 1b.8 L78150 total 305 (86.2%)  91.8%  94.2%  96% consensus prevalence Corresponding        nucleic acid database Europe  US  Rp Figure probe     Codon 72-77 amino acid n = 191/m = 25 n = 306 n = 52 n = 25 strip Acc. No AGAAAATTAGTAGATTTC PKLVDF 74w5 --------------- ----- 320 (87.9%) 264  48  16 1c.1 L78150 74w8    -----------C---  -----   9 (2.5%)  34   1   2 1c.2 L78147 74w9 -----G-----G--- -----   0  17   3   2 1c.3 L78137 74m6 ------G-------- --V--   0   5   0   3 1c.4 L78149 74m12    ------AC----  --T--   0   1   1   1 1c.5 L78136 total 329 (90.4%)  93.5%  98.1%  96% consensus prevalence Corresponding        nucleic acid database Europe  US  Rp Figure probe     Codon 182-185 amino acid n = 191/m = 25 n = 306 n = 52 n = 25 strip Acc. No CAATACATGGAT QYMD 184w11 ------------ ---- 285 (88.5%) 267  46  18 1d.1 L78147 184w17 --C--------- ----  16 (5.0%)   9   4   3 1d.2 L78137 184w21 -----T------ ----   6 (1.9%)   4   2   1 1d.3 L78145 184w24 -----------C ----   0   1   0   i 1d.4 L78144 184m12 ------G----- --V-   1 (0.3%)   8   0   1 1d.5 L78142 184m28 --------A--- --I-   3 (0.9%)   0   0   1 1d.6 L78148 total 311 (96.6%)  93.8%  98.1% 100% consensus prevalence Corresponding        nucleic acid database Europe  US  Rp Figure probe     Codon 212-218 amino acid n = 191/m = 25 n = 306 n = 52 n = 25 strip Acc. No TGGGGATTTACCACACCAGAC WGFTTPD 215w11      G------------   ----   9 (2.8%)   5   3   2 1e.1 L78146 215w9    ---------------  ----- 142 (44.2%) 178  24   3 1e.2 L78141 215w29^(f) ------------ ---- 142 105  16   3 1e.2 L78141 215w33    -----C------  ----   9 (2.8%)   8   4   1 1e.3 L78154 215w10       C-----------   L---  25 (7.8%)  10   0   2 1e.4 L78150 215w27^(f) ------C----- --L-  25  14   0   2 1e.4 L78150 215m50    --GC--------  -L--   0   0   0   1 1e.5 L78145 215w53    -----A------  -L--   1 (0.3%)   1   3   1 1e.6 L78138 215w22          --T---------    ----   3 (0.9%)  10   2   1 1e.7 L78134 215m17    ------TA----  --Y-  88 (27.4%)  50  12   7 1e.8 L78144 215m14    --G---TA----  --Y-  24 (7.5%)  24   1   1 1e.9 L78149 215m49    --G---TAT---  --Y-   0   2   0   2 1e.10 L78148 215m45       ---TT------   -F--   1 (0.3%)  16   0   1 1e.11 L78135 215m13    ---C--TA----  -LY-   0   0  16   2 1e.12 L78155 total 302 (94.1%)  92.8%  90.4%  96% consensus prevalence Corresponding        nucleic acid database Europe  US  Rp Figure probe     Codon 217-220 amino acid n = 191/m = 25 n = 306 n = 52 n = 25 strip Acc. No CCAGACAAAAAA PDKK 219m2 ------------ ---- 179 (87.7%)  26  42  18 1f.1 L78144 219m4 ------C----- --Q-   1 (0.5%)   2   4   2 1f.2 L78135 219m7 -----TC----- --Q-   0   0   0   1 1f.3 L78133 219m9 ------G----- --E-   0   0   0   1 1f.4 pending total 179 (88.2%)  82.4%  82.7%  84.6% n = amount of sequences (database retrieved), or isolates tested m = amount of motifs corresponding with the n database sequences * = redundant probes RP: reference panel The total percentage for European and US samples is not the sum of probe reactivities but a result of the complete interpretations for these codons.

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164 1 15 DNA Artificial sequence Synthetic Primer 1 agaaatggaa aagga 15 2 15 DNA Artificial sequence Synthetic Primer 2 tgtacagaaa tggaa 15 3 15 DNA Artificial sequence Synthetic Primer 3 aaatggaaaa ggaag 15 4 14 DNA Artificial sequence Synthetic Primer 4 tacagagatg gaaa 14 5 15 DNA Artificial sequence Synthetic Primer 5 gtacagagat ggaaa 15 6 15 DNA Artificial sequence Synthetic Primer 6 agagatggaa aaaga 15 7 15 DNA Artificial sequence Synthetic Primer 7 agaaatggag aagga 15 8 14 DNA Artificial sequence Synthetic Primer 8 acagagatgg aaaa 14 9 14 DNA Artificial sequence Synthetic Primer 9 gtacagagat ggaa 14 10 14 DNA Artificial sequence Synthetic Primer 10 cagagatgga aaag 14 11 15 DNA Artificial sequence Synthetic Primer 11 agaaatggaa aaaga 15 12 14 DNA Artificial sequence Synthetic Primer 12 gaaatggaaa aaga 14 13 16 DNA Artificial sequence Synthetic Primer 13 cagaaatgga aaaaga 16 14 16 DNA Artificial sequence Synthetic Primer 14 agaaatggaa aaagaa 16 15 15 DNA Artificial sequence Synthetic Primer 15 agaattggaa aagga 15 16 15 DNA Artificial sequence Synthetic Primer 16 agagttggaa aagga 15 17 14 DNA Artificial sequence Synthetic Primer 17 agagctggaa aagg 14 18 14 DNA Artificial sequence Synthetic Primer 18 agaactggaa aagg 14 19 13 DNA Artificial sequence Synthetic Primer 19 gagctggaaa agg 13 20 15 DNA Artificial sequence Synthetic Primer 20 acagaattgg aaaag 15 21 14 DNA Artificial sequence Synthetic Primer 21 acagaattgg aaaa 14 22 14 DNA Artificial sequence Synthetic Primer 22 acagaactgg aaaa 14 23 14 DNA Artificial sequence Synthetic Primer 23 agaattggaa gagg 14 24 15 DNA Artificial sequence Synthetic Primer 24 cagaattgga agagg 15 25 15 DNA Artificial sequence Synthetic Primer 25 agaattggaa gagga 15 26 14 DNA Artificial sequence Synthetic Primer 26 agaactggaa gagg 14 27 15 DNA Artificial sequence Synthetic Primer 27 cagaactgga agagg 15 28 15 DNA Artificial sequence Synthetic Primer 28 agaactggaa gagga 15 29 14 DNA Artificial sequence Synthetic Primer 29 caaaaattgg gcct 14 30 15 DNA Artificial sequence Synthetic Primer 30 atttcaagaa ttggg 15 31 14 DNA Artificial sequence Synthetic Primer 31 ttcaaaagtt gggc 14 32 15 DNA Artificial sequence Synthetic Primer 32 caaaaatcgg gcctg 15 33 15 DNA Artificial sequence Synthetic Primer 33 aaaaatcggg cctga 15 34 15 DNA Artificial sequence Synthetic Primer 34 aaagaagaaa gacag 15 35 19 DNA Artificial sequence Synthetic Primer 35 ataaagaaaa agaacagta 19 36 16 DNA Artificial sequence Synthetic Primer 36 agtactaaat ggagaa 16 37 16 DNA Artificial sequence Synthetic Primer 37 agtgataaat ggagaa 16 38 16 DNA Artificial sequence Synthetic Primer 38 acagtactaa atggag 16 39 27 DNA Artificial sequence Synthetic Primer 39 atcaggatgg agttcataac ccatcca 27 40 16 DNA Artificial sequence Synthetic Primer 40 taaatggaga aaatag 16 41 15 DNA Artificial sequence Synthetic Primer 41 gtgatagatg gagaa 15 42 14 DNA Artificial sequence Synthetic Primer 42 gtactagatg gaga 14 43 15 DNA Artificial sequence Synthetic Primer 43 agtactagat ggaga 15 44 15 DNA Artificial sequence Synthetic Primer 44 cagtaataga tggag 15 45 15 DNA Artificial sequence Synthetic Primer 45 cagtaataga tggag 15 46 15 DNA Artificial sequence Synthetic Primer 46 acagtgctag atgga 15 47 14 DNA Artificial sequence Synthetic Primer 47 cagtgctaga tgga 14 48 14 DNA Artificial sequence Synthetic Primer 48 cagtgctaga tgga 14 49 14 DNA Artificial sequence Synthetic Primer 49 cagtgataga tgga 14 50 15 DNA Artificial sequence Synthetic Primer 50 cagtgataga tggag 15 51 14 DNA Artificial sequence Synthetic Primer 51 agtgatagat ggag 14 52 15 DNA Artificial sequence Synthetic Primer 52 agtgatagat ggaga 15 53 18 DNA Artificial sequence Synthetic Primer 53 gagaaaatta gtagattt 18 54 16 DNA Artificial sequence Synthetic Primer 54 aaaattagta gacttc 16 55 17 DNA Artificial sequence Synthetic Primer 55 gagaaagtta gtggatt 17 56 17 DNA Artificial sequence Synthetic Primer 56 agaaaagtag tagattt 17 57 16 DNA Artificial sequence Synthetic Primer 57 aaaattaaca gatttc 16 58 16 DNA Artificial sequence Synthetic Primer 58 gaaaattaac agattt 16 59 17 DNA Artificial sequence Synthetic Primer 59 gaaaattaac agatttc 17 60 15 DNA Artificial sequence Synthetic Primer 60 cttccacagg gatgg 15 61 15 DNA Artificial sequence Synthetic Primer 61 cttccacaag gatgg 15 62 16 DNA Artificial sequence Synthetic Primer 62 tgctcccaca gggatg 16 63 15 DNA Artificial sequence Synthetic Primer 63 cttccaatgg gatgg 15 64 16 DNA Artificial sequence Synthetic Primer 64 gcttccaatg ggatgg 16 65 18 DNA Artificial sequence Synthetic Primer 65 agttatctat caatacag 18 66 16 DNA Artificial sequence Synthetic Primer 66 agttatctgt caatac 16 67 17 DNA Artificial sequence Synthetic Primer 67 tcaatacatg gatgagg 17 68 17 DNA Artificial sequence Synthetic Primer 68 tcagtacatg gatgagg 17 69 16 DNA Artificial sequence Synthetic Primer 69 atcaatacat ggatga 16 70 14 DNA Artificial sequence Synthetic Primer 70 tcagtacatg gatg 14 71 15 DNA Artificial sequence Synthetic Primer 71 atcaatatat ggatg 15 72 16 DNA Artificial sequence Synthetic Primer 72 atcaatatat ggatga 16 73 15 DNA Artificial sequence Synthetic Primer 73 tcaatatatg gatga 15 74 15 DNA Artificial sequence Synthetic Primer 74 tcaatacatg gacga 15 75 15 DNA Artificial sequence Synthetic Primer 75 caatacatgg acgat 15 76 16 DNA Artificial sequence Synthetic Primer 76 tcaatacatg gacgat 16 77 17 DNA Artificial sequence Synthetic Primer 77 caatacgtgg atgaggg 17 78 14 DNA Artificial sequence Synthetic Primer 78 aatacataga tgat 14 79 15 DNA Artificial sequence Synthetic Primer 79 caatacatag atgat 15 80 16 DNA Artificial sequence Synthetic Primer 80 caatacatag atgatt 16 81 15 DNA Artificial sequence Synthetic Primer 81 caatacgtag atgat 15 82 15 DNA Artificial sequence Synthetic Primer 82 tcaatacgtg gatga 15 83 16 DNA Artificial sequence Synthetic Primer 83 tcaatacata gatgat 16 84 17 DNA Artificial sequence Synthetic Primer 84 atcaatacat agatgat 17 85 15 DNA Artificial sequence Synthetic Primer 85 ggatttacca cacca 15 86 14 DNA Artificial sequence Synthetic Primer 86 gacttaccac acca 14 87 14 DNA Artificial sequence Synthetic Primer 87 ggtttaccac acca 14 88 14 DNA Artificial sequence Synthetic Primer 88 gatttaccac acca 14 89 14 DNA Artificial sequence Synthetic Primer 89 ttactacacc agac 14 90 13 DNA Artificial sequence Synthetic Primer 90 ttaccacacc aga 13 91 14 DNA Artificial sequence Synthetic Primer 91 tggggactta ccac 14 92 14 DNA Artificial sequence Synthetic Primer 92 tggggattta ccac 14 93 14 DNA Artificial sequence Synthetic Primer 93 ggggttcacc acac 14 94 17 DNA Artificial sequence Synthetic Primer 94 gggatttacc acaccag 17 95 17 DNA Artificial sequence Synthetic Primer 95 gggatttacc acaccag 17 96 17 DNA Artificial sequence Synthetic Primer 96 tggggactta ccacacc 17 97 17 DNA Artificial sequence Synthetic Primer 97 tgggggttta ccacacc 17 98 17 DNA Artificial sequence Synthetic Primer 98 gggatttact acaccag 17 99 14 DNA Artificial sequence Synthetic Primer 99 gggattaacc acac 14 100 14 DNA Artificial sequence Synthetic Primer 100 ggggattaac caca 14 101 15 DNA Artificial sequence Synthetic Primer 101 tggggattaa ccaca 15 102 14 DNA Artificial sequence Synthetic Primer 102 gggggttaac caca 14 103 14 DNA Artificial sequence Synthetic Primer 103 ggggttaacc acac 14 104 14 DNA Artificial sequence Synthetic Primer 104 tgggggttaa ccac 14 105 14 DNA Artificial sequence Synthetic Primer 105 gggattgacc acac 14 106 14 DNA Artificial sequence Synthetic Primer 106 ggattgacca cacc 14 107 13 DNA Artificial sequence Synthetic Primer 107 gggattgacc aca 13 108 13 DNA Artificial sequence Synthetic Primer 108 gggactgacc aca 13 109 14 DNA Artificial sequence Synthetic Primer 109 gggactgacc acac 14 110 15 DNA Artificial sequence Synthetic Primer 110 tgggggttaa ccaca 15 111 15 DNA Artificial sequence Synthetic Primer 111 tgtggttaac cccca 15 112 14 DNA Artificial sequence Synthetic Primer 112 ggggcttacc acac 14 113 14 DNA Artificial sequence Synthetic Primer 113 ggactttaca cacc 14 114 14 DNA Artificial sequence Synthetic Primer 114 gggttttaca cacc 14 115 15 DNA Artificial sequence Synthetic Primer 115 ggatttttca cacca 15 116 14 DNA Artificial sequence Synthetic Primer 116 ggattttaca cacc 14 117 17 DNA Artificial sequence Synthetic Primer 117 gggattttac acaccag 17 118 17 DNA Artificial sequence Synthetic Primer 118 gggatttttc acaccag 17 119 14 DNA Artificial sequence Synthetic Primer 119 gggattttac acac 14 120 14 DNA Artificial sequence Synthetic Primer 120 ggggatttta caca 14 121 14 DNA Artificial sequence Synthetic Primer 121 ccctaaaatg tgtg 14 122 14 DNA Artificial sequence Synthetic Primer 122 ggatttttca cacc 14 123 14 DNA Artificial sequence Synthetic Primer 123 gatttttcac acca 14 124 14 DNA Artificial sequence Synthetic Primer 124 gggatttttc acac 14 125 14 DNA Artificial sequence Synthetic Primer 125 cccctaaaat gtgt 14 126 14 DNA Artificial sequence Synthetic Primer 126 ggttttatac acca 14 127 14 DNA Artificial sequence Synthetic Primer 127 gggttttata cacc 14 128 14 DNA Artificial sequence Synthetic Primer 128 ggggttttat acac 14 129 14 DNA Artificial sequence Synthetic Primer 129 gggggcttac caca 14 130 14 DNA Artificial sequence Synthetic Primer 130 ggattctaca cacc 14 131 13 DNA Artificial sequence Synthetic Primer 131 gattctacac acc 13 132 13 DNA Artificial sequence Synthetic Primer 132 ggattctaca cac 13 133 14 DNA Artificial sequence Synthetic Primer 133 gggattctac acac 14 134 14 DNA Artificial sequence Synthetic Primer 134 gggttttata cccc 14 135 13 DNA Artificial sequence Synthetic Primer 135 ggttttatac ccc 13 136 13 DNA Artificial sequence Synthetic Primer 136 gttttatacc cca 13 137 15 DNA Artificial sequence Synthetic Primer 137 accagacaaa aaaca 15 138 14 DNA Artificial sequence Synthetic Primer 138 gggactgacc acac 14 139 15 DNA Artificial sequence Synthetic Primer 139 caccagacaa aaaac 15 140 14 DNA Artificial sequence Synthetic Primer 140 cagacaagaa acat 14 141 14 DNA Artificial sequence Synthetic Primer 141 ccagacaaga aaca 14 142 15 DNA Artificial sequence Synthetic Primer 142 accagacaag aaaca 15 143 14 DNA Artificial sequence Synthetic Primer 143 agacaaaaag catc 14 144 14 DNA Artificial sequence Synthetic Primer 144 cagacaaaaa gcat 14 145 15 DNA Artificial sequence Synthetic Primer 145 cagacaaaaa gcatc 15 146 14 DNA Artificial sequence Synthetic Primer 146 ccagataaaa aaca 14 147 14 DNA Artificial sequence Synthetic Primer 147 accagataaa aaac 14 148 15 DNA Artificial sequence Synthetic Primer 148 cccagataaa aaaca 15 149 16 DNA Artificial sequence Synthetic Primer 149 ccagataaaa aacatc 16 150 15 DNA Artificial sequence Synthetic Primer 150 caccagataa aaaac 15 151 15 DNA Artificial sequence Synthetic Primer 151 cagacaagaa acatc 15 152 14 DNA Artificial sequence Synthetic Primer 152 accagacaag aaac 14 153 15 DNA Artificial sequence Synthetic Primer 153 accagaccaa aaaca 15 154 15 DNA Artificial sequence Synthetic Primer 154 accagacgaa aaaca 15 155 15 DNA Artificial sequence Synthetic Primer 155 accagatcaa aaaca 15 156 14 DNA Artificial sequence Synthetic Primer 156 accagatcaa aaac 14 157 15 DNA Artificial sequence Synthetic Primer 157 caccagatca aaaac 15 158 14 DNA Artificial sequence Synthetic Primer 158 accagacgaa aaac 14 159 14 DNA Artificial sequence Synthetic Primer 159 ccagacgaaa aaca 14 160 14 DNA Artificial sequence Synthetic Primer 160 ccagaccaaa aaca 14 161 14 DNA Artificial sequence Synthetic Primer 161 accagaccaa aaac 14 162 29 DNA Artificial sequence Synthetic Primer 162 gtacagtatt agtaggacct acacctgtc 29 163 29 DNA Artificial sequence Synthetic Primer 163 ccaaaagtta aacaatggcc attgacaga 29 164 20 DNA Artificial sequence Synthetic Primer 164 agttcataac ccatccaaag 20 

What is claimed is:
 1. Method for determining the susceptibility to antiviral drugs of viruses which contain reverse transcriptase genes and are present in a biological sample, comprising: (i) hybridizing polynucleic acids present in the sample with at least two reverse transcriptase (RT) gene probes, with said probes being applied to known locations on a solid support and with said probes being capable of simultaneously hybridizing to their respective target regions under appropriate hybridization and wash conditions allowing the detection of homologous targets, or with said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence wherein T of said target sequence is replaced by U; (ii) detecting the hybrids formed in step (i); and (iii) inferring the nucleotide sequence at the codons of interest as represented in any of FIG. 1, or Table 4 and/or the amino acids of the codons of interest and/or antiviral drug resistance spectrum from the differential hybridization signal(s) obtained in step (ii); wherein said viruses are HIV strains, and wherein said RT gene probes are each selected from the group consisting of SEQ ID NO: 4, 8, 9, 10, 11, 12, 13, 14, 15, 19, 21, 22, 24, 25, 27, 28, 30, 31, 32, 33, 34, 35, 40, 46, 48, 49, 50, 51, 52, 57, 58, 70, 72, 73, 75, 76, 78, 79, 80, 81, 82, 83, 86, 88, 90, 93, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 115, 117, 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 157, and
 159. 2. Method according to claim 1, wherein at least one of the probes of step (i) is selected from the group consisting of SEQ ID NO: 4, 8, 9, 10, 11, 12, 13, 14, 15, 19, 21, 22, 24, 25, 27, and
 28. 3. Method according to claim 1, wherein at least one of the probes of step (i) is selected from the group consisting of SEQ ID NO: 30, 31, 32, and
 33. 4. Method according to claim 1, wherein at least one of the probes of step (i) is selected from the group consisting of SEQ ID NO: 34, 35, 40, 46, 48, 49, 50, 51, and
 52. 5. Method according to claim 1, wherein at least one of the probes of step (i) is selected from the group consisting of SEQ ID NO: 57, and
 58. 6. Method according to claim 1, wherein at least one of the probes of step (i) is selected from the group consisting of SEQ ID NO: 70, 72, 73, 75, 76, 78, 79, 80, 81, 82, and
 83. 7. Method according to claim 1, wherein at least one of the probes of step (i) is selected from the group consisting of SEQ ID NO: 86, 88, 90, 93, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 115, 117, 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, and
 136. 8. Method according to claim 1, wherein at least one of the probes of step (i) is selected from the group consisting of SEQ ID NO: 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 157, and
 159. 9. At least two probes on a solid support; wherein the probes comprise at least one first probe selected from the group consisting of SEQ ID NO: 4, 8, 9, 10, 11, 12, 13, 14, 15, 19, 21, 22, 24, 25, 27, 28, 30, 31, 32, 33, 34, 35, 40, 46, 48, 49, 50, 51, 52, 57, 58, 70, 72, 73, 75, 76, 78, 79, 80, 81, 82, 83, 86, 88, 90, 93, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 115, 117, 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 157, and 159; and at least one other second probe selected from the group consisting of SEQ ID NO: 4, 8, 9, 10, 11, 12, 13, 14, 15, 19, 21, 22, 24, 25, 27, 28, 30, 31, 32, 33, 34, 35, 40, 46, 48, 49, 50, 51, 52, 57, 58, 70, 72, 73, 75, 76, 78, 79, 80, 81, 82, 83, 86, 88, 90, 93, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 115, 117, 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 157, and
 159. 10. Composition comprising at least one first probe selected from the group consisting of SEQ ID NO: 4, 8, 9, 10, 11, 12, 13, 14, 15, 19, 21, 22, 24, 25, 27, 28, 30, 31, 32, 33, 34, 35, 40, 46, 48, 49, 50, 51, 52, 57, 58, 70, 72, 73, 75, 76, 78, 79, 80, 81, 82, 83, 86, 88, 90, 93, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 115, 117, 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 157, and 159; and at least one other second probe selected from the group consisting of SEQ ID NO: 4, 8, 9, 10, 11, 12, 13, 14, 15, 19, 21,22, 24, 25, 27, 28, 30, 31, 32, 33, 34, 35, 40, 46, 48, 49, 50, 51, 52, 57, 58, 70, 72, 73, 75, 76, 78, 79, 80, 81, 82, 83, 86, 88, 90, 93, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 115, 117, 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 157, and
 159. 